Method and apparatus for isolating and purifying nucleic acid using a single surface

ABSTRACT

Provided is a method of isolating nucleic acid from cells using a single surface, wherein a compound represented by Formula 1 is bound to the surface. Also provided are an apparatus for isolation of nucleic acids, and a bead for isolating nucleic acids.

This application claims the benefit of Korean Patent Application No.10-2006-0092921, filed on Sep. 25, 2006, and all the benefits accruingtherefrom under 35 U.S.C. §119, the disclosure of which is incorporatedherein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and apparatus for isolatingand purifying nucleic acid using a single surface, and moreparticularly, a method of performing cell concentration, cell disruptionand DNA purification using a single surface.

2. Description of the Related Art

In general, to extract DNA from a clinical sample, processes ofconcentrating target cells in a sample, disrupting the concentratedcells, and isolating and/or purifying DNA released from the disruptedcells have to be performed. To perform such processes on a solidsupport, surfaces having different chemical functional groups suitablefor each operation have to be used. However, in general, a surfaceuseful for cell concentration and a surface useful for DNA purificationrequired different functional groups. Therefore, in order to use suchsurfaces having different functional groups suitable for each operationin a lab-on-a-chip (“LOC”), separate chambers having surfacesappropriate to each operation need to be manufactured, resulting in acomplicated system and method of manufacture. In addition, a pluralityof pumps and valves are required to flow fluids into each chamber,further complicating the system. Therefore, there is a need for a singlesurface that can both bind cells in a clinical sample and DNAs releasedfrom the sample.

U.S. Pat. No. 6,617,105 discloses a method of isolating nucleic acidfrom a sample containing cells, the method comprising: binding cells inthe sample to a solid support coated with a cell binding moiety;disrupting the isolated cell; binding nucleic acid released from thedisrupted cells to the solid support; and recovering the nucleic acidfrom the solid support. However, in U.S. Pat. No. 6,617,105, chaotropicsalts or detergents are used for cell disruption, and a method disclosedin U.S. Pat. No. 5,234,809 is used for nucleic acid purification. U.S.Pat. No. 5,234,809 discloses a process for isolating nucleic acids froma nucleic acid-containing starting material, comprising mixing thestarting material, a chaotropic substance and a nucleic acid bindingsolid phase, separating the solid phase with the nucleic acid boundthereto from the liquid, and washing the solid phase nucleic acidcomplexes. Therefore, the inventors of the present invention haveearnestly studied to solve the problems of the prior art, and found thata single surface comprising a compound having a hydrophobic moiety forcell separation and a pH dependent charge switching moiety for nucleicacid purification permits efficient isolation of cells and subsequentisolation of nucleic acids released from the cells, thus completing thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides a method of isolating nucleic acid from acell using a single surface to which a compound having a hydrophobicmoiety for cell separation and a pH dependent charge switching moietyfor nucleic acid purification is bound, wherein cell concentration, celldisruption and nucleic acid purification can be efficiently performed onthe single surface. The method comprises: mixing a sample containingcells with a solution comprising beads dispersed in a binding buffer tobind the cells to the beads; separating the beads having the cells boundthereto from the binding buffer and then washing the beads with a washbuffer; disrupting the cells to release nucleic acids to bind the beads;and eluting bound nucleic acid from the beads using an elution buffer,wherein a compound represented by Formula 1 below is bound to thesurface of the beads:

where Z₁ is a carboxyl group or an amino group;

R₁, R₂, R₃, R₅, R₆, R₇, R₈, R₉ and R₁₁ are each independently selectedfrom the group consisting of a hydrogen atom, a halogen atom, a hydroxylgroup, a substituted or unsubstituted C1-C20 alkyl group, a substitutedor unsubstituted C1-C20 alkoxy group, a substituted or unsubstitutedC2-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,and a substituted or unsubstituted C6-C30 aryloxy group;

R₄ is a substituted or unsubstituted C4-C20 alkyl group, a substitutedor unsubstituted C4-C20 alkoxy group, a substituted or unsubstitutedC4-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,or a substituted or unsubstituted C6-C30 aryloxy group;

R₁₀ is a nitrogen-containing heteroaryl or heterocyclic group of 3-30carbon atoms;

j, k, l and m are each independently an integer in the range of 1-10;and

n is an integer in the range of 1-30,000.

The present invention also provides an apparatus for isolating nucleicacid from a cell using a single surface to which a compound having ahydrophobic moiety for cell separation and a pH dependent chargeswitching moiety for nucleic acid purification is bound, wherein cellconcentration, cell disruption and nucleic acid purification can beefficiently performed on the single surface. The apparatus comprises acell disruption micro chamber having a sample inlet through which asample is introduced; a bead dispersion storage part in fluidcommunication with the cell disruption micro chamber which supplies abead dispersion to the micro chamber; a binding buffer storage part influid communication with the cell or virus disruption micro chamberwhich supplies a binding buffer to the micro chamber; a nucleic acideluting buffer storage part in fluid communication with the celldisruption micro chamber which supplies a nucleic acid eluting buffer tothe micro chamber; and a laser generation part attached to the celldisruption micro chamber which irradiates the micro chamber with alaser.

The present invention also provides a lab-on-a-chip including theapparatus.

The present invention also provides a bead for isolating nucleic acidfrom a cell, wherein a compound represented by Formula 1 is bound to thesurface of the bead.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1 illustrates a bead used in the method according to an embodimentof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to an embodiment of the present invention, there is provided amethod of isolating and purifying nucleic acid from cells, the methodcomprising: mixing a sample containing cells with a solution comprisingbeads dispersed in a binding buffer to bind the cells onto the beads;separating the beads having the cells bound thereto from the bindingbuffer and then washing the beads with a wash buffer; disrupting thebound cells to release nucleic acid such that the nucleic acid binds tothe beads; optionally washing the beads having nucleic acid boundthereto with the wash buffer; and eluting bound nucleic acid from thebeads using an elution buffer, wherein a compound represented by Formula1 below is bound to the surface of the beads:

where Z₁ is a carboxyl group or an amino group;

R₁, R₂, R₃, R₅, R₆, R₇, R₈, R₉ and R₁₁ are each independently selectedfrom the group consisting of a hydrogen atom, a halogen atom, a hydroxylgroup, a substituted or unsubstituted C1-C20 alkyl group, a substitutedor unsubstituted C1-C20 alkoxy group, a substituted or unsubstitutedC2-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,and a substituted or unsubstituted C6-C30 aryloxy group;

R₄ is a substituted or unsubstituted C4-C20 alkyl group, a substitutedor unsubstituted C4-C20 alkoxy group, a substituted or unsubstitutedC4-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,or a substituted or unsubstituted C6-C30 aryloxy group;

R₁₀ is a nitrogen-containing heteroaryl or a heterocyclic group of 3-30carbon atoms;

j, k, l and m are each independently an integer in the range of 1-10;and

n is an integer in the range of 1-30,000. As used herein, the term“cell” means a prokaryotic or eukaryotic cell, a plant cell, a bacteriacell, a pathogenic cell, a yeast cell, an aggregate of cells, a virus, afungus, or other nucleic acid containing biological material, such as,for example, an organelle.

In an embodiment, the beads used in the method have a surface that has ahydrophobic functional group that can bind cells in a clinical sampleand a functional moiety that can bind DNA released from the cells underthe same conditions as that for cell binding but which can release thebound DNA when the pH changes. In Formula 1, R₄ is a functional groupcorresponding to the hydrophobic moiety that can bind cells, andrepresents a hydrocarbon of at least 4 carbon atoms, preferably, asubstituted or unsubstituted C4-C20 alkyl group, a substituted orunsubstituted C4-C20 alkoxy group, a substituted or unsubstituted C4-C20alkenyl group, a substituted or unsubstituted C6-C30 aryl group, or asubstituted or unsubstituted C6-C30 aryloxy group. In the currentembodiment, when a sample containing cells is mixed with a solutioncomprising beads dispersed in a binding buffer, the cells are bound tothe hydrophobic moiety of the compound of Formula I bound to the bead.

When the concentration of the cells in a sample is low because thevolume of the sample is large, after the cells are bound to a bead, thebead can be separated from the sample. The bead can be separated fromthe sample by applying a magnet or an electromagnetic field to thesample, or by centrifugation or the like, but the method of separatingthe beads is not limited thereto. After separation from the sample, thebeads can be dispersed in the binding buffer, or in any other buffersuitable for lysing the cells. At this time, the volume of buffer intowhich the beads are placed can be very small, for example, about 5 μl.

Cells bound to the dispersed beads are disrupted by laser-irradiation orheating. For disrupting the cells, heating can be performed at 94-96° C.for 3-10 minutes.

When laser-irradiation is used to disrupt the bound cells, beads thatabsorb a laser beam, for example, magnetic beads, can be used.Laser-irradiation of a solution containing magnetic beads causes cellablation on the beads, due to the transfer of shock waves, vaporpressure and heat to the cell surfaces as a result of the energy of thelaser beam. At the same time, physical shocks are also applied to thecell surfaces. The magnetic beads are heated by the laser, raise thetemperature of the solution, and directly disrupt the cells. Themagnetic beads in the solution do not act simply as a heat conductor;but in addition they apply thermal, mechanical and physical shocks tothe cell surface, thereby efficiently disrupting the cells.

The term “magnetic bead” as used herein means that the bead is capableof having a magnetic moment imparted to it when placed in a magneticfield, and thus is displaceable under the action of that field.

The laser beam used to irradiate the beads can be generated by a pulselaser or a continuous wave (CW) laser. The laser power is about 10 mW ormore for the CW laser and about 1 mJ/pulse or more for the pulse laser.Preferably, the pulse laser has a power of about 3 mJ/pulse or more andthe CW laser has a power of about 100 mW or more. Laser ablation cannoteffectively occur if the laser power is too low. When the power of theCW laser is less than about 10 mW or the power of the pulse laser isless than about 1 mJ/pulse, insufficient energy to disrupt the cells istransferred.

The laser beam is generated in a specific wavelength range in whichmagnetic beads can absorb the energy of the laser. The laser beam isgenerated preferably in the wavelength range of about 400 nm or higher,and more preferably in the wavelength range from about 750 nm to about1,300 nm. In an embodiment, the laser beam is generated in a wavelengthrange in which DNA is not damaged or denatured. DNA can be denatured ordamaged when the wavelength of the laser is less than about 400 nm. Thelaser beam can also be generated in one or more wavelengths. That is,the laser can have one wavelength or two or more different wavelengthswithin the above range.

The diameter of the magnetic beads is preferably from about 50 nm toabout 1,000 μm, and more preferably from about 1 μm to about 50 μm. Whenthe diameter of the magnetic beads is less than about 50 nm, physicaland mechanical shocks are insufficient to cause cell lysis. When thediameter of the magnetic beads is greater than about 1,000 μm, it is notsuitable for LOC. The magnetic beads can also be a mixture of beads withtwo or more sizes. That is, the magnetic beads can be of the same sizeor can be a mixture of beads of different sizes.

When cells are disrupted, for example by irradiation with a laser beam,nucleic acids are released from the cells. The nucleic acids are boundto the beads via the DNA binding moiety of the compound represented byFormula I bound to the beads. In Formula 1, R₁₀ is a functional groupcorresponding to a DNA binding moiety. R₁₀ is a nitrogen-containingheteroaryl or heterocyclic group of C3-C30; preferably, it is apyridinyl group or an imidazolyl group.

Subsequently, the beads having nucleic acid bound thereto can be washedwith a wash buffer. As a result, impurities that are not bound to thebead, for example, cell debris or proteins can be removed. The washbuffer can be the binding buffer, or any other buffer suitable forremoval of the impurities without removing the bound nucleic acid.

To elute purified nucleic acid, a nucleic acid elution buffer can beadded to the beads Irradiating the beads with a laser beam or heatingthe beads can be simultaneously performed with addition of the elutionbuffer to increase the efficiency of eluting the bound nucleic acid fromthe beads.

The surface of the bead has a compound represented by Formula 1 boundthereto. In a compound according to an embodiment of the presentinvention, a carboxyl group or an amino group represented by Z₁, can bebound to the bead by a peptide bond.

In Formula 1 above, R₁ and R₅ can each be a hydroxyl group. When R₁ andR₅ are each a hydroxyl group, they are each a part of a carboxylic acidwhich can function as a nucleic acid eluting moiety, depending on pH.

FIG. 1 illustrates a bead used in the method according to an embodimentof the present invention. In FIG. 1, the bead is a magnetic bead, andwithin the charge-switching moiety of the compound of Formula 1, a DNAbinding moiety is an imidazolyl group, and a DNA releasing moiety is acarboxyl group.

According to an embodiment of the present invention, R₁, R₂, R₃, R₅, R₆,R₇, R₈, R₉ and R₁₁ of the compound represented by Formula I can eachcomprise an alkyl group that has a straight or branched radical ofC1-C20, preferably, a straight or branched radical of C1-C12.Preferably, the alkyl radical is a lower alkyl radical having 1-6 carbonatoms. Examples of the alkyl radical include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, amyl, iso-amyl, hexylor the like. More preferably, the alkyl radical is a lower alkyl radicalhaving 1-3 carbon atoms.

According to an embodiment of the present invention, R₁, R₂, R₃, R₅, R₆,R₇, R₈, R₉ and R₁₁ of the compound represented by Formula I can eachcomprise an alkoxy group that is an oxygen-containing straight orbranched radical including an alkyl part of C1-C20. Preferably, thealkoxy radical is a lower radical having 1-6 carbon atoms. Examples ofthe alkoxy radical include methoxy, ethoxy, propoxy, butoxy andt-butoxy. More preferably, the alkoxy radical is a lower alkoxy radicalhaving 1-3 carbon atoms. The alkoxy radical can be a haloalkoxy radicalthat is substituted with at least one halo atom such as fluoro, chloroor bromo. More preferably, the haloalkoxy radical is a lower haloalkoxyradical having 1-3 carbon atoms. Examples of the haloalkoxy radicalinclude fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy,fluoroethoxy and fluoropropoxy.

According to an embodiment of the present invention, R₁, R₂, R₃, R₅, R₆,R₇, R₈, R₉ and R₁₁ of the compound represented by Formula I can eachcomprise an alkenyl group that has a carbon-carbon double bond and aC2-C30 straight or branched aliphatic hydrocarbon. The alkenyl group maypreferably have 2-12 carbon atoms within a ring, more preferably, 2-6carbon atoms within a ring. The term “branched” represents that at leastone lower alkyl group or lower alkenyl group is attached to the straightchain of the alkenyl group. The alkenyl group can be unsubstituted, orcan be independently substituted by at least one functional group whichincludes halo, carboxy, hydroxyl, fomyl, sulfo, sulfino, carbamoyl,amino and imino, but is not limited thereto. Examples of the alkenylgroup include ethenyl, prophenyl, carboxyethenyl, carboxyprophenyl,sulfinoethenyl and sulfonoethenyl.

The aryl group as used herein, which is used alone or in combination,refers to a carbocyclic aromatic system of 6-20 carbon atoms having oneor more rings. The rings may be attached to each other as a pendantgroup or may be fused. The term “aryl” includes an aromatic radical suchas phenyl, naphthyl, tetrahydronaphthyl, indane, and biphenyl. Phenyl ismore preferable. The aryl group may have 1-3 substituents such ashydroxy, halo, haloalkyl, nitro, cyano, alkoxy, and lower alkylamino.

The aryloxy group as used herein refers to aryl-O—. The definition ofthe “aryl” in the aryloxy group is as described above.

In the method according to an embodiment of the present invention, thebinding buffer can have a pH of about 3 to about 5. When the pH of thebinding buffer is beyond this range, binding efficiency between cells ornucleic acids and the beads is decreased. The binding buffer can bephosphate buffer, acetate buffer, citrate buffer, MES buffer or thelike, but is not limited thereto. The binding buffer may have aconcentration of about 10 to about 1,000 mM. When the concentration ofthe binding buffer is less than the lower end of this range, efficiencyof binding of cells or nucleic acids to the beads and efficiency ofwashing impurities, such as cell debris or proteins, from the beads arereduced. When the concentration of the binding buffer is greater thanthe higher end of this range, preparation of the binding buffer isdifficult.

In the method according to an embodiment of the present invention, thenucleic acid elution buffer may have a pH of about 8 to about 10. Whenthe pH of the nucleic acid elution buffer is less than the lower end ofthis range, eluting efficiency of nucleic acid is reduced. When the pHof the nucleic acid elution buffer is greater than the higher end ofthis range, subsequent processes can be affected. The nucleic acidelution buffer can be phosphate buffer, HEPES buffer, Tris buffer or thelike, but is not limited thereto. The nucleic acid elution buffer mayhave a concentration of about 10 to about 1,000 mM. When theconcentration of the nucleic acid elution buffer is outside this range,eluting efficiency of nucleic acid from the beads is reduced, andsubsequent processes can be also affected.

In the method according to an embodiment of the present invention, thebead can be a magnetic bead, a silica bead, a polystyrene bead, a glassbead, a cellulose bead or the like, but is not limited thereto.Preferably, the bead is a magnetic bead when laser-irradiation is usedfor disrupting cells bound to the beads, since magnetic beads can absorbenergy from the laser. On the other hand, when beads that do not absorbenergy from a laser are used, for example, a silica bead, a polystyrenebead, a glass bead, or a cellulose bead, disruption of cells can beperformed by heating.

In an embodiment of the present invention, the sample containing cellscan be saliva, urine, blood, serum, a cell culture or the like, but isnot limited thereto. The sample can be any solution comprising nucleicacids, such as animal cells, plant cells, bacteria, viruses, phage andthe like.

According to another embodiment of the present invention, there isprovided an apparatus for continuously performing isolation andpurification of nucleic acid, the apparatus comprising: a celldisruption micro chamber having a sample inlet through which a samplecontaining cells is introduced; a bead dispersion storing part in fluidcommunication with the cell disruption micro chamber through a microchannel which supplies a bead dispersion to the micro chamber throughthe micro channel; a binding buffer storing part in fluid communicationwith the cell disruption micro chamber through a micro channel whichsupplies a binding buffer to the micro chamber through the microchannel; a nucleic acid elution buffer storing part in fluidcommunication with the cell disruption micro chamber through a microchannel which supplies a nucleic acid elution buffer to the microchamber through the micro channel; and a laser generation part attachedto the cell disruption micro chamber which supplies a laser beam toirradiate the micro chamber.

The micro chamber that disrupts cells includes an inlet through which asample comprising cells is introduced. The sample is thoroughly mixedwith beads, a process that can be achieved by various mixing methods,for example vibration. A laser beam can irradiate the mixture of thesample and the beads while vibrating the mixture. A cell disruptionchamber window should be composed of a material transparent to the laserbeam. Magnetic beads exposed to the laser transform light to heat, i.e.laser ablation. Heat, vibration, shock wave, vapor pressure, etc. areefficiently transferred to the cells due to effective heat transfer andcollision of the magnetic beads with bound cells by continuousvibration.

The vibrator used to generate the vibration can be a sonicator, avibrator using a magnetic field, a vibrator using an electric field, amechanical vibrator such as a vortex etc., or a piezoelectric material.A vibrator is attached to the micro chamber and can be any devicecapable of vibrating the solution of the cells and the beads.

In an embodiment, the apparatus can further comprise an electromagnetthat is attached to the micro chamber. The electromagnet is attached tothe micro chamber to immobilize magnetic beads in a predetermined spaceof the micro chamber. When a sample containing cells flows over theimmobilized magnetic beads in the micro chamber, the cells can bind tothe magnetic beads.

In the absence of the electromagnet, extra steps have to be taken toseparate the beads having cells bound thereto from the sample. Forexample, magnetic beads and a sample containing cells are mixed to bindthe cells to the magnetic beads, the magnetic beads are next separatedfrom the sample, and dispersed in a buffer. When an electromagnet isused, the separation process described above becomes unnecessary, andthe number of steps of separating cells can be reduced. For the purposeof LOC implementation, the magnetic beads can be recovered orconstrained to one position in the micro chamber by applying anelectromagnet field after disruption of the cells. In so doing, nucleicacids released from the cells can be purified without a step ofseparating the magnetic beads from the sample, and therefore the elutedpurified nucleic acid solution can directly feed into a chamber for asubsequent use, such as a Polymerase Chain Reaction (“PCR”) chamber.

The apparatus according to an embodiment of the present invention canfurther include a nucleic acid amplification chamber in fluidcommunication with the micro chamber through a microchannel. For thepurpose of the LOC implementation, an amplification system of thepurified nucleic acid is necessary. The purified nucleic acid can bedetected using a spectrophotometer, an electrochemical method,electrochemiluminescence, radiation or fluorescent labeling, a real-timePCR method, or the like. The PCR method is most suitable to sufficientlyamplify a desired DNA. Other DNA amplification methods can be appliedand direct detection through the real-time PCR method, etc. is alsopossible.

The laser generating part can generate a pulse laser or a continuouswave (CW) laser. The laser power is about 10 mW or more for the CW laserand about 1 mJ/pulse or more for the pulse laser. Preferably, the powerof the pulse laser is about 3 mJ/pulse or more and the power of the CWlaser is about 100 mW or more.

The laser should be generated in a specific wavelength range at whichmagnetic beads absorb the energy of the laser. The laser is generatedpreferably in the wavelength range of about 400 nm or more, and morepreferably in the wavelength range from about 750 nm to about 1,300 nm.The laser can also be generated in multiple wavelengths. That is, thelaser can have one wavelength or two or more wavelengths within theabove range.

According to another embodiment of the present invention, there isprovided a lab-on-a-chip comprising the apparatus for continuouslyperforming nucleic acid isolation and purification. Each functionalelement of the apparatus for isolating and purifying nucleic acid can bedesigned for a process-on-a-chip, furthermore, the lab-on-a-chip can bedesigned using known microfluidic techniques and MEMS devices.

According to another embodiment of the present invention, there isprovided a bead for isolating and purifying nucleic acid from cells,wherein a compound represented by Formula 1 below is bound to thesurface of the bead.

where Z₁ is a carboxyl group or an amino group;

R₁, R₂, R₃, R₅, R₆, R₇, R₈, R₉ and R₁₁ are each independently selectedfrom the group consisting of a hydrogen atom, a halogen atom, a hydroxylgroup, a substituted or unsubstituted C1-C20 alkyl group, a substitutedor unsubstituted C1-C20 alkoxy group, a substituted or unsubstitutedC2-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,and a substituted or unsubstituted C6-C30 aryloxy group;

R₄ is a substituted or unsubstituted C4-C20 alkyl group, a substitutedor unsubstituted C4-C20 alkoxy group, a substituted or unsubstitutedC4-C20 alkenyl group, a substituted or unsubstituted C6-C30 aryl group,or a substituted or unsubstituted C6-C30 aryloxy group;

R₁₀ is a nitrogen-containing heteroaryl or a heterocyclic group of 3-30carbon atoms;

j, k, l and m are each independently an integer in the range of 1-10;and

n is an integer in the range of 1-30,000.

In the bead according to the current embodiment of the presentinvention, R₁₀ can be a pyridinyl group or an imidazolyl group, and eachof R₁ and R₅ can be a hydroxyl group.

The present invention will now be described in greater detail withreference to the following examples. The following examples are forillustrative purposes only and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Bead Surface Treatment

1) Coupling of Polyanhydrides (A Functional Group was Introduced forIntroducing a Hydrophobic Functional Group and an Ionizable FunctionalGroup)

1 ml of magnetic beads having an amine functional group, DYNABEADS M-270Amine (Invitrogen), was washed with 1 ml of N-methyl-2-pyrolidone (NMP)three times. The magnetic beads were separated from the solution using amagnet, and then 1 ml of 200 mM (based on a repeating unit)polyanhydride (poly(isobutyl-alt-maleic anhydride)) having an averagemolecular weight of 50,000, dissolved in NMP, was added to the magneticbeads. Thereafter, the beads and the polyanhydride were mixed for onehour to obtain the resulting product and then washed with NMP threetimes.

2) Introduction of a Positive Ionizable Group

The solvent was removed from the magnetic bead solution of process 1).Then, 1 ml of a solution of 3 mM 1-(3-aminopropyl)imidazole and 6 mMtriethylamine dissolved in NMP was added to the magnetic beads. Thebeads and the solution were mixed for one hour, and then washed with NMPthree times.

3) Introduction of a Negative Ionizable Group

The solvent was removed from the resulting magnetic bead solution ofprocess 2). 1 ml of a 0.01 N NaOH solution was added to the magneticbeads. Then, the beads and the NaOH solution were mixed for 30 minutesand washed with 1 ml of distilled water five times. Lastly, theresulting product was dispersed in 1 ml of sterilized triple distilledwater and cold stored (4° C.).

Example 2 Cell Concentration Using the Method According to an Embodimentof the Present Invention

50 μl of the solution comprising beads dispersed in distilled water(prepared in Example 1) was washed twice with 100 μl of a cell bindingbuffer (100 mM sodium acetate, pH 4.0) and then dispersed in 100 μl ofthe same buffer. 10 μl of E. coli BL21 (OD₆₀₀=1.0) in 1× phosphatebuffered saline (PBS) was mixed with 40 μl of a 1×PBS buffer, 40 μl ofurine, or 40 μl of blood, respectively. Then, each of the resultingsamples was mixed with a prepared bead solution for two minutes.Subsequently, the beads were separated from the sample using a magnet. Asample of each cell solution, without mixing with the beads, was diluted1/10,000 in 1×PBS. Similarly, each cell solution recovered after mixingwith the beads was diluted 1/10,000 in 1×PBS. Then, an aliquot of thediluted cell solutions was plated on 3 M Petri film and the number ofcolonies were counted to calculate binding efficiency. The results areshown below in Table 1.

TABLE 1 Sample PBS buffer Urine Blood Binding Efficiency >99% >99% 25.0± 5.7%

As can be seen in Table 1, in the PBS buffer and urine, E. coli cellswere efficiently bound to the beads. However, binding efficiency of EColi cells to the beads in blood to was relatively low. It is consideredthat this is because a relatively large amount of material exists in theblood inhibits E. coli cells from binding to the beads.

Example 3 Cell Disruption and Nucleic Acid Purification Using the MethodAccording to an Embodiment of the Present Invention

The beads bound with E. coli cells of Example 2 were dispersed in 5 μlof a binding buffer, and the resulting solution was placed in amicrochip for a TMC-1000 (Samsung Techwin). Then, the bead solution wasirradiated with a laser (Hamamatsu 8446-72, 1.5 W, 808 nm) for 40seconds. The bead solution was then removed from the microchip toquantify DNA using PICOGREEN. The beads resulting from the aboveprocedures were dispersed in 5 μl of the binding buffer (100 mM sodiumacetate buffer, pH 4.0), and mixed for one minute. Then, the beads wereremoved from the binding buffer and dispersed in 5 μl of an elutionbuffer (100 mM Tris, pH 9.5). The resulting solution of beads in theelution buffer was placed in a microchip for a TMC-1000 (SamsungTechwin). The solution was irradiated for 40 seconds with a 1.5 W laser,and then the elution buffer was collected to quantify the eluted DNAusing PICOGREEN. The results of quantification of DNA existing in thesolutions before DNA elution (after cell disruption) and after DNAelution are shown in Table 2 below.

TABLE 2 Sample PBS buffer Urine After cell disruption <0.1 ng/μl <0.1ng/μl After DNA elution 6.1 ± 1.1 ng/μl 5.0 ± 0.9 ng/μl

As can be seen in Table 2, since little DNA exists in the binding bufferbefore nucleic acids are eluted from the beads, most of the DNA is boundto the beads after cell disruption. In addition, it can be seen that alarge amount of DNA is recovered from the beads using the elutionbuffer.

Therefore, it is confirmed that after cells bound to beads aredisrupted, nucleic acids released from the cells can be eluted from thebeads using a buffer that has a different pH from that of the bindingbuffer.

Example 4 Purity Measurement of the Purified Nucleic Acid Using theMethod According to an Embodiment of the Present Invention

Values of A₂₆₀/A₂₈₀ for the eluted DNA solution of Example 3 weremeasured using a NANODROP (NanoDrop Technology). An experiment wasperformed as a control in which only cell disruption was performedwithout nucleic acid purification. 5 μl of a 1×PBS buffer containing E.coli (OD₆₀₀=2.0) was added to 100 μl of beads (Dynabead® MyOne™carboxylic acid; Invitrogen). The bead-cell mixture was irradiated for40 seconds with a laser beam, and then the buffer solution was separatedfrom the beads prior to measuring A₂₆₀/A₂₈₀ in order to preventinterference of the beads in the spectroscopic measurements. Values ofA₂₆₀/A₂₈₀ with respect to samples of the present invention in which DNApurification was performed after cell disruption and samples of thecontrol in which DNA purification was not performed are shown in Table 3below.

TABLE 3 Sample Example 3 sample Control A₂₆₀/A₂₈₀ 1.92 ± 0.10 2.74 ±0.34

As can be seen in Table 3, a sample obtained using the method of thepresent invention has a value of A₂₆₀/A₂₈₀ in the range of 1.8-2.0.Therefore, it can be seen that the sample obtained using the method ofthe present invention is purer than the control in which no nucleic acidpurification process was performed.

Example 5 Real-Time PCR Using Nucleic Acid Purified by the MethodAccording to an Embodiment of the Present Invention

To additionally confirm the effect of DNA purification, a real-time PCRwas performed using the DNA solution prepared in Example 3. A PCR mastermixture was prepared containing 2×PCR buffer (150 mM Tris-HCl (pH 9.0),30 mM Ammonium sulfate, 5 mM gCl₂, and 0.2 mg/ml Bovine Serum Albumin(“BSA”); Solgent, Korea), 0.6 U/μl of Taq polymerase (Solgent), 0.4 mMof dNTP, 0.4 μM of a forward primer, 0.4 μM of a reverse primer, 10 mMof MgCl₂, and 2× (relative to what concentration is this fluorophore2×?) SYBR-green. Then, the PCR master mixture was mixed with the DNAsolution prepared in Example 3 in a ratio of 1:1, and PCR was performedusing the TMC-1000. Conditions of PCR were as follows: initialdenaturation at 94° C. for one minute, and then 40 cycles of 94° C. for5 seconds, 62° C. for 5 seconds, and 72° C. for 40 seconds. The regionto be amplified is the 16s rRNA gene, and the primer sequences used areas follows.

Forward primer: 5′-YCC AKA CTC CTA CGG GAG GC-3′ (SEQ ID NO: 1)

Reverse primer: 5′-GTA TTA CCG CTT CTG CTG GCA C-3′ (SEQ ID NO: 2)

Table 4 below represents results of real-time PCR performed using DNAswith or without DNA purification as measured by Ct. Ct represents thecycle number at which a fluorescence signal became detectable in thereal-time PCR. That is, the higher the initial concentration of DNA inthe reaction sample used for the PCR, the earlier the fluorescencesignal can be detected in the real time PCR (i.e. fluorescence signalscan be detected at a lower Ct value). Ct is also related to the purityof the DNA used in the real time PCR. That is, the purer the DNA, thelower the Ct value.

TABLE 4 Ct for Sample Ct for Urine PBS buffer PresentInvention(purified) 19.86 ± 0.59 23.92 ± 0.32 Control(not purified)27.08 ± 1.54 33.86 ± 0.88

As can be seen in Table 4, samples of the present invention in which DNApurification is performed have much lower Ct values than those ofsamples of the control regardless of sample type, due to the effect ofDNA purification using the method of the present invention.

Therefore, it is confirmed that DNA subjected to the method of thepresent invention is purer than DNA in a control sample which was notsubjected to the method of the present invention.

After PCR was completed, the solution was removed from the chip toanalyze the PCR product using a LabChip (Aglient). Table 5 representsresults of real-time PCR using DNA purified (by the method of thepresent invention) and unpurified (control) DNA.

TABLE 5 Sample Urine PBS buffer Present 13.9 ± 1.7 ng/μl  11.9 ± 3.7ng/μl  Invention(purified) Control(not purified) 7.8 ± 1.7 ng/μl 1.9 ±0.5 ng/μl

As can be seen in Table 5, samples of the present invention in which DNAwas purified produced much more amplicon compared with samples of acontrol regardless of sample type.

According to the method of the present invention, cell concentration,cell disruption and DNA purification can be performed using the samesurface that can bind cells and DNAs under the same condition. Since thewhole process is performed using the same beads, a system can be easilymanufactured, and can be also widely used in CDs, microchips and thelike.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item. Theterm “or” means “and/or”. The terms “comprising”, “having”, “including”,and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to”).

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The endpoints of all ranges are includedwithin the range and independently combinable.

All methods described herein can be performed in a suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (e.g., “suchas”), is intended merely to better illustrate the invention and does notpose a limitation on the scope of the invention unless otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention as used herein. Unless defined otherwise, technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in the art to which this invention belongs.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method of isolating nucleic acid from cells, the method comprising:mixing a sample containing cells with a solution comprising beadsdispersed in a binding buffer to bind the cells to the beads; separatingthe beads having the cells bound thereto from the binding buffer andthen washing the beads with a wash buffer; disrupting the bound cells torelease nucleic acid to bind to the beads; and eluting bound nucleicacid from the beads using an elution buffer, wherein a compoundrepresented by Formula 1 below is bound to the surface of the beads:

where Z₁ is a carboxyl group or an amino group; R₁, R₂, R₃, R₅, R₆, R₇,R₈, R₉ and R₁₁ are each independently selected from the group consistingof a hydrogen atom, a halogen atom, a hydroxyl group, a substituted orunsubstituted C1-C20 alkyl group, a substituted or unsubstituted C1-C20alkoxy group, a substituted or unsubstituted C2-C20 alkenyl group, asubstituted or unsubstituted C6-C30 aryl group, and a substituted orunsubstituted C6-C30 aryloxy group; R₄ is a substituted or unsubstitutedC4-C20 alkyl group, a substituted or unsubstituted C4-C20 alkoxy group,a substituted or unsubstituted C4-C20 alkenyl group, a substituted orunsubstituted C6-C30 aryl group, or a substituted or unsubstitutedC6-C30 aryloxy group; R₁₀ is a nitrogen-containing heteroaryl orheterocyclic group of 3-30 carbon atoms; j, k, l and m are eachindependently an integer in the range of 1-10; and n is an integer inthe range of 1-30,000.
 2. The method of claim 1, wherein the disruptingof the cells is performed by heating or by laser-irradiation.
 3. Themethod of claim 2, wherein the laser is a pulse laser or a continuouswave (CW) laser.
 4. The method of claim 3, wherein the pulse laser has apower of at least about 3 mJ/pulse, and the CW laser has a power of atleast about 100 mW.
 5. The method of claim 3, wherein the wavelength ofthe laser beam is at least about 400 nm.
 6. The method of claim 1,wherein the binding buffer has a pH of about 3 to about
 5. 7. The methodof claim 1, wherein the elution buffer has a pH of about 8 to about 10.8. The method of claim 1, wherein eluting bound nucleic acid from thebeads is performed simultaneously with irradiating the beads with alaser or heating.
 9. The method of claim 1, wherein the beads aremagnetic beads, silica beads, polystyrene beads, glass beads, orcellulose beads.
 10. The method of claim 1, wherein R₁₀ is a pyridinylgroup or an imidazolyl group.
 11. The method of claim 1, wherein R₁ andR₅ are each a hydroxyl group.
 12. The method of claim 1, wherein thesample is saliva, urine, blood, serum, or a cell culture.